Neuritic plaques containing primarily amyloid beta protein (Abeta or Aβ) are one of the hallmarks of Alzheimer's Disease. Beta-site APP cleaving enzyme (BACE), known also as beta-secretase, β-secretase, Asp2, and Memapsin, has been identified as the enzyme responsible for processing amyloid precursor protein (APP) to produce the N-terminal portion of the Abeta peptide. This enzyme has been suggested as rate limiting in the production of the Abeta peptide. See, for example, Sinha et al., 1999, Nature 402:537-554, and published PCT applications WO 00/17369, WO 01/23533, and WO 00/47618. See also: Hussain, I. et al., 1999, Mol. Cell. Neurosci. 14:419-427; Vassar, R. et al., 1999, Science 286:735-741; Yan, R. et al., 2000, Nature 402:533-537; and Lin, X. et al., 2000, Proc. Natl. Acad. Sci. USA 97:1456-1460 (2000).
BACE is a relatively large and structurally complex enzyme. The primary structure of BACE as it is synthesized in the endoplasmic reticulum is shown in FIG. 1. The enzyme contains 501 amino acids, including a N-terminal signal (leader) sequence of about 21 amino acids (pre-sequence domain) followed by a pro-sequence domain consisting approximately of residues 22 to 45 (pro-sequence domain) that is proteolytically removed once the enzyme reaches its destination in the Golgi apparatus, to generate a mature enzyme.
BACE contains a transmembrane domain of about 27 amino acids that anchors the protein to the membrane. A short cytosolic C-terminal tail of 21 amino acids follows the transmembrane domain. Attachment to the membrane allows BACE to interact with and cleave APP, the first and prerequisite step in the generation of A-beta.
BACE isolated from human brain is heavily glycosylated. As expressed by a stably transfected 293T cell line, BACE is glycosylated at four asparagines: 132, 151, 202, and 333. Analysis of HEK 293 cells stably overexpressing BACE showed that the enzyme is phosphorylated at Ser477, and that phosphorylation regulates enzyme intracellular trafficing (Walter et. al., 2001, J. Biol. Chem. 276:14634-41). Three disulfide bonds suggested as critical for activity, are formed between the following pairs of cysteine residues: Cys216-Cys420, Cys278-Cys443, and Cys330-Cys380 (Haniu et. al., 2000, J. Biol. Chem. 275:21099-21106).
These structural features of the BACE polypeptide all appear to have specific functions relating to enzymatic activity. Enzymes expressed in insect and CHO cells are properly refolded and show activity. These proteins are glycosylated. For example, insect cells express glycosylated BACE from the mannose-rich glycans available in the insect cells. Biantennary and triantennary oligosaccharides of the complex type provide glycosylation in the CHO-expressed BACE (Charlwood et. al., 2001 J. Biol. Chem. 276:16739-48).
Published patent applications WO 00/47618, WO 01/23533 and WO 00/17369 identify the beta-secretase enzyme and various methods of its use. To better understand the mechanism of action of β-secretase and help explore novel strategies for drug discovery for Alzheimer's disease, it has become important to understand the role of the four N-linked glycosylation sites of BACE. This understanding will make it possible to explore mutations in BACE that enzyme activity as well as explore potential active sites for target molecules.